This action might not be possible to undo. Are you sure you want to continue?
Ryan Kerneya,1, Eunsoo Kimb, Roger P. Hangarterc, Aaron A. Heissa, Cory D. Bishopd, and Brian K. Halla
a Department of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1; bDepartment of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5; cDepartment of Biology, Indiana University, Bloomington, IN 47405; and dDepartment of Biology, St. Francis Xavier University, Antigonish, NS, Canada B2G 2W5
Edited by David B. Wake, University of California, Berkeley, CA, and approved February 18, 2011 (received for review December 6, 2010)
The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae (“Oophila amblystomatis” Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA ampliﬁcation reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26–44 through chlorophyll autoﬂuorescence and algal 18S rDNA ampliﬁcation. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae through FISH, suggesting that the decline in autoﬂuorescent cells is primarily due to algal cell death within the host. However, early embryonic egg capsules also contained encysted algal cells on the inner capsule wall, and algal 18S rDNA was ampliﬁed from adult reproductive tracts, consistent with oviductal transmission of algae from one salamander generation to the next. The invasion of algae into salamander host tissues and cells represents a unique association between a vertebrate and a eukaryotic alga, with implications for research into cell–cell recognition, possible exchange of metabolites or DNA, and potential congruence between host and symbiont population structures.
| endosymbiosis | amphibian | chlorophyte
utualistic endosymbiosis occurs in many protist and even metazoan groups. One of the most prominent forms of endosymbiosis is between photosynthetic microbes and eukaryotic hosts. Photosynthetic endosymbionts and plastids are hosted by many eukaryote lineages, including several invertebrate animals (1). However, despite the ability of pathogenic organisms to enter vertebrate cells (2), there is an apparent absence of mutualist endosymbionts in vertebrates (3). The inability of symbionts to enter vertebrate host cells may be attributable to the adaptive immune system (3), a gnathostome synapomorphy that recognizes and destroys foreign cells (4). Associations between microbes and vertebrate embryos are possible candidates for intracellular symbioses because such early associations may precede an adaptive immune response. The symbiosis between algae and embryos of Ambystoma maculatum (spotted salamander) was ﬁrst reported more than 120 y ago (5). These salamanders spend most of their adult lives underground but emerge for semiannual spring breeding congregations in vernal pools. Eggs are deposited in these pools, where embryos develop through metamorphosis. Once transformed, the young salamanders leave these pools to live underground. Algae live in direct association with embryos inside salamander egg capsules, which are contained in large jelly masses (6). Individual egg capsules appear green due to dense accumulations of algae surrounding the embryo (Fig. 1A). The mutual beneﬁt of this facultative association has been clearly established through exclusion experiments. Clutches raised in the dark do not accrue detectable algae (6–9). The presence of algae in these experiments correlates with earlier hatching (6, 9), decreased embryonic mortality (7, 8), more synchronous hatching
(8, 9), and reaching a larger size (8) and later developmental stage (9) at hatching. Additionally, algal growth is minimal in egg capsules after embryos are removed (8), indicating that the embryos, and not the egg capsules, aid algal growth. Algae are thought to beneﬁt from nitrogenous wastes released by the embryos [ref. 6; supported by research in the closely related Ambystoma gracilis (10)], whereas salamander embryos beneﬁt from increased oxygen concentrations associated with the algae [refs. 11–13; however, others (14) found no oxygen beneﬁt using older techniques]. Presence of the algae also correlates with decreased cilium-mediated rotation of early-stage embryos and increased muscular contractions during later embryonic stages (9), both of which may be secondary effects of modulated oxygen levels. Despite these physiological studies, structural and developmental aspects of this symbiosis remain unclear. Early attempts to identify or culture the algae from oviducts of gravid female salamanders failed, discouraging further investigations into the anatomical associations between host and symbiont (8, 15). All previous research describes the relationship between the algae and embryo as an ectosymbiotic mutualism, with no intratissue or intracellular stages. In this study we show that algae invade embryonic salamander tissues (Figs. 1 and 2) and cells (Figs. 3 and 4) during embryonic development. This intracellular invasion resembles the photosynthetic endosymbiosis that has occurred in many protists (16) and metazoan invertebrates (1, 17, 18) but has not been reported in a vertebrate host. Results Phylogenetic reconstruction, using chloroplast 16S (1,362 bp; GenBank accession no. HM590633) and nuclear 18S (1,714 bp; GenBank accession no. HM590634) rDNA sequences from intracapsular algae, places the algae in the Chlorophyceae, speciﬁcally the Chlamydomonadales (Fig. S1). Two algal clones of each PCR ampliﬁcation had >99% sequence identity to each other, and the 16S and 18S rDNA phylogenies were congruent. These observations suggest that intracapsular algal cells in our samples comprise a single species. The published literature on this association (6, 8, 9) refers to the algae as “Oophila amblystomatis” (Lambert ex Printz) (19) on the basis of an informal species designation by Lambert (6), although—to date—no formal taxonomic description exists. Previous studies reported
Author contributions: R.K. designed research; R.K., E.K., R.P.H., A.A.H., and C.D.B. performed research; E.K., R.P.H., A.A.H., and C.D.B. contributed new reagents/analytic tools; R.K. and B.K.H. analyzed data; and R.K., E.K., R.P.H., A.A.H., C.D.B., and B.K.H. wrote the paper. The authors declare no conﬂict of interest. This article is a PNAS Direct Submission. Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. HM590633 (Oophila sp. 16S) and HM590634 (Oophila sp. 18S)].
To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1018259108/-/DCSupplemental.
PNAS | April 19, 2011 | vol. 108 | no. 16 | 6497–6502
adjacent to the blastopore (arrows). He. 2. (G) Stage-46 liver.1018259108 Intratissue algae were ﬁrst detected through their chlorophyll autoﬂuorescence (480 nm excitation) in a Stage-39 embryo (Fig.Fig. Janthinobacterium sp. However. C. (A and B) Coronal and (C–F) transverse vibratome sections imaged for chlorophyll autoﬂuorescence. Autoﬂuorescent cells were scattered throughout the embryo. S2).) additional bacteria and protists within the egg capsules (6. Algal cells from the egg capsule invade salamander tissues. and ﬂuorescence microscopy. It was not detected in Stage-17 (neurula). 6498 | www. 50 μm in E. algae were detected microscopically on the innermost egg capsule during these early stages (Fig. Individual algae were embedded in (A) the otic capsule (Ot). Verrucomicrobiales sp. embryos (Fig. (Scale bars: 1 mm in A–D. Boxed region in D shown magniﬁed in E. or earlier. and older. lateral view of the head. branchial arch.. and Flavobacterium sp. (C) Black-and-white and ﬂuorescent overlay of a Stage-39 embryo removed from its egg capsule. 14). Acidovorax sp.1073/pnas. Algal cell entry into host tissues was veriﬁed through PCR ampliﬁcation of symbiont 18S rDNA (20). (B) the pharynx (Ph) and pharyngeal clefts. Gilbert (6) described a concentration of algae around the salamander blastopore (“proctodeum”). (Scale bars: 100 μm in A and D.. (B) Still frame taken from timelapse recordings of Stage-15 embryos.. Nt. time-lapse photography. heart. . hyoid arch. Ph. Concentrations of algae occur synchronously. Time-lapse photography of A. The timing of algal cell entry was determined through algal 18S rDNA PCR. Oophila-speciﬁc 18S rDNA was ampliﬁed and sequenced from Harrison (21) Stage-26 (pharyngula). salamander embryos. (A) A Stage-44 embryo inside its egg capsule (Ec). Duganella zoogloeoides. and several regions of the anterior (E) and posterior trunk (F).org/cgi/doi/10. 1C). 1. with a high concentration in the anterior end of the ventral abdomen. 1 mm in B. (F–H) FISH-stained algal cells embedded in (F) Stage-37 cranial mesenchyme.pnas. E. (D and E) Scattered algal cells embedded in Stage-37 cranial mesenchyme appear green under light microscopy. Red spots are autoﬂuorescing cells (A) described in text. (C and D) several regions of the head. However. neural tube. and our 16S rDNA ampliﬁcation clones included sequences for the prokaryotes Pedobacter sp. including the alimentary canal (Al) and notochord (No). Our 18S rDNA ampliﬁcation clones included sequences from ciliate and cercozoan protists. Arrows indicate autoﬂuorescent algal cells. and F. Chitinophaga sp. Hy. Ba. Boxed region in C shown in higher magniﬁcation in D. Distribution of algal cells in salamander embryos.. no other green algae were identiﬁed from the egg capsules aside from the conserved Oophila sequences. maculatum embryos revealed this concentration forming in Kerney et al.) Fig. 20 μm in F–H. pharynx. Embryos were extracted from their egg capsules and rinsed thoroughly before DNA extraction. and (H) Stage-46 ceratohyal chondrocyte.. including the neural tube (Nt). 3 A and B).
heart. (C and D) TEM images of intracapsular algae revealing starch granules (S). adjacent to salamander endoderm. neural tube. Algal cells within the alimentary canal during the process of cellular invasion. indicating that the algal bloom outside the blastopore precedes subsequent algal cell invasion into the salamander embryo host. yolk platelet. F. (Scale bars: 100 μm in A. (F–J) Intracellular algae found within somites. and otic capsule between Stages 35 and 44 (Figs. 1 mm in E. (E) Coronal vibratome section through the trunk region of a Stage-37 embryo. FISH-positive cells were found in the larval liver (n = 2. in comparison with the widespread distribution of algae during embryonic Stages 35–44. Fig. notochord. 3. we identiﬁed algal cells in embryonic epidermis. (B) TEM image of algal cells encased by an outer envelope (En. nucleus. Yp.) Stage-15 embryos (Fig. Many of the intratissue autoﬂuorescing cells appeared green under light microscopy in vibratome sections (Fig. (A) Fluorescent image of a coronal vibratome section through a Stage-35 embryo showing an aggregation of algae (A) inside the alimentary canal (Al) and invading the surrounding endoderm. boxed region in 3A) on the inner border of the innermost egg capsule. (I) Algal cell surrounded by a thickened outer envelope. FISH-positive algal cells were detected in several Stage-37 tissues. somitic myotome. cranial mesenchyme. 5 μm in D. Autoﬂuorescing cells were no longer detectable in feeding larvae (Stage 46. (E) Algal cell inside endodermal cell of the alimentary canal containing many large vacuoles (V) and paired ﬂagella (F). 108 | no. Fig. Nt. No. neural tube. chloroplast grana (G). (F) Algal cell embedded inside a salamander myocyte (M). 3 to 4 wk of development). 2011 | vol. This concentration of algal cells precedes their detection inside salamander tissues through PCR. 1B and Movie S1). 2 μm in F–J. (G) Higher magniﬁcation of intracellular alga in F. White spots are autoﬂuorescing cells embedded in the somites (So). 2 μm in B and C. yolk platelet. (J) Degraded algal cell without an outer envelope. and vacuoles (V). Autoﬂuorescing cells were more abundant in the yolk and within the alimentary canal (Fig. most algae persist in the intracapsular ﬂuid and are released into the surrounding water upon hatching. lipid droplet. 1H) after the cessation of detectable algal autoﬂuorescence. Kerney et al. 16 | 6499 DEVELOPMENTAL BIOLOGY . presumptive lens. (A) Whole-mount image of egg capsule (Ec) showing clusters of algal cells. Intracapsular and intracellular algae. optic cup. and 4A). nuclei (N). Yp.) Fig. 1 μm in F. LD. The genetic identity of intratissue algae was veriﬁed through ﬂuorescence in situ hybridization (FISH) using an oligonucleotide probe that targets Oophila 18S rRNA. (F) Free thylakoid membranes (T) and starch granules (S) inside cytoplasm of host cell shown in E. 4A). By imaging chlorophyll autoﬂuorescence.Fig. By Stage 46. (B–F) TEM images from boxed region in A. N. 2 μm in E. We detected few algal cells in our Stage-46 sample through FISH. 1 D and E). Transmission electron microscopy (TEM) was used to determine the ultrastructure of algae attached to the egg capsules PNAS | April 19. 2 μm in B–D. 3E. Intratissue autoﬂuorescent cells and algae from the intracapsular ﬂuid both ﬂuoresced red under the same excitation wavelength (480 nm). (Scale bars: 1 mm in A. Although many algal cells invaded the alimentary canal and surrounding tissues. 2. including the cranial mesenchyme (Fig. (H) Paired algal cells within a host cell. 1G) and ceratohyal cartilage (n = 1. 1F) and endoderm. 4. ﬂagella. Salamander mitochondria (Mi) and myoﬁbrils (My) occur in the surrounding host cytoplasm. (B–D) Algal cells in the alimentary canal. H.
4E). poriferan. Recent research has also revealed lateral gene transfer of algal nuclear genes into the sea slug E. and foraminiferan hosts (1. 8.05]. motile algae within the egg capsule ﬂuid (Fig. Fig.70. 3 F–I) and embedded within endodermal cells surrounding the alimentary canal (n = 13.. Previous research on invertebrate–alga associations provides ready-made experimental hypotheses for the Ambystoma–alga symbiosis. see ref. Vacuoles reached a peak size in algae Kerney et al. some scattered intracellular algae found in A. This resembles endosymbioses between cyanobacterial Prochloron sp. Algal cells within the alimentary canal occurred in loose aggregates (Fig. and between the green alga Chlorella and ciliate. Such experiments will require careful attention to the occurrence of photosynthate transfer vs. Elysia chlorotica) from their algal food (e. In the wild.0% SD 15%) algae [F(2.05]. chloroplast grana. Fig. Fig. Structural Changes to Invading Algae. The absence of detectable mitosis or cytokinesis in any of the singular or paired intracellular algae suggests that these paired cells invaded the salamander host cell together. 3G). 4 B–D). often adjacent to the boundary between two endodermal cells.3% SD 13%). 3B). Although most intracellular algal cells appeared intact (Fig. Algae on the egg capsules were encased in an outer envelope (Fig. 3B). 3 G–I).2%) than intracellular (5. However. 4F). 3 F–I). Unlike the case in many invertebrate photosymbionts.2%) or alimentary canal (3.5% SD 9.g. 37. unlike E. These cells were recessed from the outer envelope walls. 3 C and D). chlorotica kleptoplasts.g. 4 B–D). maculatum has not been detected (27). algal cell digestion within the host. intracellular (n = 24). There were no differences in starch granule sizes [one-way ANOVA. starch granules constituted more crosssectional area of intracapsular algae (10. 25). 3 G–J). vacuoles constituted increasing cross-sectional area of intracapsular (11. Algae found both within the alimentary canal and inside host cells had a proportional increase in vacuoles and decrease in starch granules compared with intracapsular algae. . 3I) similar to the envelope of encysted algae on the innermost egg capsule wall of early-stage embryos (Fig. dinoﬂagellates of the genus Symbiodinium and cnidarian.0% SD 8. 23).org/cgi/doi/10. chlorotica (29).1073/pnas.65) = 135. Because amphibian nuclei readily acquire foreign DNA from the cytoplasm (31).05]. P < 0. and posterior ovary (one of three) but was not found in the medial oviduct. and algae embedded in salamander tissues (Figs. nearly all intratissue algal–animal symbioses are obligatory and consist of photosynthate transfer from symbiont to host (17. 3 G–I). maculatum embryos may have transferred heritable algal DNA. poriferan. there is no obvious role for transferred algal genes in the A. and didemnid ascidian hosts (24). Discussion The invasion of green algae into salamander cells reveals that intracellular symbiosis of a phototroph can occur in a vertebrate host. maculatum genome. In adult male salamanders. All host somite cells were undergoing myocyte differentiation. Surprisingly. The latter are undoubtedly in short supply within the opaque tissues of a primarily fossorial salamander species.pnas. Many intracellular algae that did not have an outer envelope appeared degraded (e. Both intracapsular and invaded algae had paired ﬂagella (Fig. or TEM imaging. cells. Host cells showed no signs of cellular necrosis or apoptosis and were structurally similar to adjacent.89. Salamander endoderm adjacent to algal cells in the alimentary canal had indistinct plasma membranes and dissociated cytoplasm in regions of host–symbiont cellular contact. which may be associated with maintaining kleptoplast function (however.g. Algal cells were initially present in small clusters on individual egg capsules of early embryos (Fig. many autoﬂuorescent algal cells were embedded within the cytoplasm of differentiated salamander host cells. and numerous thylakoid membranes (e. However. Each intratissue alga contained starch granules inside the chloroplast. anterior oviduct (one of three). Most intracellular algae occurred as single cells. 15) and may not result in photosynthate transfer (28).2%) algae [one-way ANOVA. with vacuolar contents recessed and occasionally extruded from the cell. 27). Oophila-speciﬁc 18S rDNA was ampliﬁed from the Wolfﬁan (one of three) and Müllerian (one of three) ducts but not from the anterior or posterior testes (Fig. Algal endosymbionts. 3H). 3 C and D).5%). These perisymbiont zones were not associated with vesicle membranes. although these were apparent in only a few intracellular algae.1018259108 Nine salamander cells containing algae also contained small collections of acellular thylakoid membranes and starch granules within their cytoplasm (Fig.g.4% SD 3.. S2).56) = 2. 3J).(Fig.18. These free thylakoids were more common in endodermal cells (n = 7) than in somitic cells (n = 2). 3B) and lacked ﬂagella. 4E). However kleptoplasts are often enclosed in distinct vesicular membranes (17. Vaucheria litorea) (23). They superﬁcially resemble the intracellular kleptoplasts acquired by some sacoglossan mollusks (e. 4D).. the Ambystoma–alga symbiosis is unique in several features. photosynthate transfer from algae to A. FISH. often have a wide range of ultrastructural variation (32). previous research has shown that the Ambystoma–alga association is facultative (6. Fig. the negative result of this earlier research is not conclusive (10). Algal– animal associations are typically limited by the availability of carbon dioxide and photons. although three pairs of intracellular algae were also observed (Fig. suggesting no distinct host symbiosome surrounding each alga (20). however. and cnidarian hosts (26). 30). P > 0. several were degraded (Fig. Oophila-speciﬁc 18S rDNA was ampliﬁed from the posterior oviduct (two of three). Our data on this symbiosis justiﬁes experimentally readdressing the possibility of photosynthate transfer by tracing labeled carbon isotopes. but did not. However. In female salamanders. Intracellular algae often occurred in close proximity to host cell mitochondria (Fig. 6500 | www. Additionally. and extracellular algae found within the embryonic alimentary canal (n = 18). Relative area measurements of starch granules and vacuoles were compared between intracapsular (n = 26). We extracted DNA from reproductive tissues of three adult female and three adult male A. platyhelminth. F(2. intracellular (48. P < 0. 3A). and 42 (e. 3 F–J and 4E). Several changes to algal cells were associated with host tissue and cellular invasion.. allowing ultrastructural analysis of 26 algal cells inside salamander tissues during Stages 35. Fig. and alimentary canal (56.g. apparent through myoﬁbril formation within the cytoplasm (Fig. Five intracellular algae were encased in an outer envelope (Fig. alga-free. algae within the alimentary canal (Fig.9% SD 4. which were also found in intracapsular algae (Fig. Vibratome sections containing autoﬂuorescent cells were dissected and prepared for TEM. Algae were not found in the alimentary canal of Stage-46 larvae either through autoﬂuorescence. This variation also was apparent in our ultrastructural analyses of Oophila sp. These conserved ultrastructural features also verify the green algal identity of intratissue autoﬂuorescent cells (22).. consistent with an active process of cellular invasion (Fig. 3 G–I) and occasionally bordered host cell nuclei. However. anterior or medial ovary. maculatum. F(2.56) = 4. Two reoccurring features of intracellular photosymbionts include photosynthate transfer and lateral gene transfer. Intracellular algae were found in autoﬂuorescent somitic cells (n = 11. 3J). The methods used to detect carbon ﬁxed by photosynthesis (27) should have revealed algal cells invading host tissues. mollusk. Several structural changes correlate with alimentary canal and cellular invasion. An electron-translucent “perisymbiont zone” (20) surrounded several intracellular algae (Fig.. whereas the free thylakoids in salamander cells are not. such as Chlorella sp.
This is similar to the association between Platymonas convolutae (a prasinophycean green alga) and acoel ﬂatworms of the genus Convoluta sp.gov/ij/). A structurally similar envelope surrounds algae found on the innermost egg capsule of early-stage embryos (before tissue invasion. Additionally.found within the alimentary canal and often appeared extruded from individual algal cells (Fig. (a trebouxiophycean green alga) with cnidarian hydras (26). the lack of other intracellular symbiont examples in vertebrates may simply be due to a lack of investigation. but we know of no other observation for vertebrates (3). and imaged on an FEI Tecnai-12 transmission electron microscope. Additional algae from the intracapsular ﬂuid (Fig. and imaged for ﬂuorescence on a Zeiss Axio Observer Z1 compound microscope. Therefore. 108 | no. maculatum. Most intracellular algae. programmed cell death (33). The unexpected intracellular association shown in our study may reveal a beneﬁt to the embryo other than oxygen production. pH 7.4). 27). could serve as the behavioral cue for tissue and cellular invasion against this light gradient. This evidence against vertical transmission has led to a general acceptance that the algae are derived from the environment (34). evolution. nor did we ﬁnd algae concentrated in the reproductive tracts of embryos or early-stage larvae. which confers a meaningful physiological beneﬁt (13). The intracellular symbiosis described here reveals unanticipated complexity in the Ambystoma–alga system with implications for the ecology. However. 4D). Tissue regions containing bright autoﬂuorescing cells were manually dissected from vibratome sections. Possibility of Vertical Transmission. Polymerized resin blocks were trimmed and sectioned to a thickness of 70 nm. or even algal cell encystment (22. Materials and Methods Animal Care. and algae of the alimentary canal. ﬁxed overnight at 4 °C in Karnovsky’s solution (2% paraformaldehyde. Previous research using light microscopy did not ﬁnd algae in the oviducts of A. The combination of an embryonic symbiosis and an inefﬁcient immune system may. Their possible absorption may confer a metabolic beneﬁt to their host. The process of algal cell integration in the Ambystoma–alga symbiosis differs from that described in other animal–alga symbioses. disappear by early larval stages. in part. account for the acceptance of an intracellular symbiont in A. The vacuoles of intracapsular green algae in Ambystoma gracile hosts have been shown to be sites of ammonia waste storage in the form of proteins (10). previous studies have had difﬁculty in obtaining alga-free clutches from either the laboratory or the wild (6. maculatum (15) and failed to culture algae from oviduct (6. 18. 15). Invertebrate hosts typically ingest their algal symbionts. and population growth to the algae. Hutchison and Hammen (14) found no net gain of oxygen from the algae and instead attributed the algal contribution to unknown “growth factors” supplied to the embryo. 10). Mixed modes of vertical and horizontal acquisition of symbionts are common.1 M cacodylate buffer (30 min). 1A) were aspirated with a 32-gauge needle and processed in parallel to the salamander tissues. The reciprocal beneﬁts of hatchling survival and growth to the embryo. consistent with algae having entered salamander embryos directly by penetrating their embryonic integument. PNAS | April 19. these vacuoles also resemble the accumulation bodies of encysting dinoﬂagellates. Intracellular symbionts have been reported for many metazoan taxa. All image area measurements were made with Image J (http://rsbweb.and xenografts (37). maculatum. However. which are a form of autophagic vesicle (22). some algal cells are embedded within epithelial or mesenchymal tissues that are far from the alimentary canal. indicating that they may be involved in a process of algal cell encystment after invasion. counterstained with Reynold’s lead citrate. Endosymbiosis. 2011 | vol.1 M sodium cacodylate buffer. 16 | 6501 DEVELOPMENTAL BIOLOGY . Fig. Additionally. The ampliﬁcation of algal 18S rDNA from the oviducts. Embryo clutches were collected from the Halifax Regional Municipality. the increase of vacuole size may be associated with alimentary canal and cellular invasion. Similar phylogenetic Kerney et al. Algal cells leave direct sunlight by entering opaque salamander tissues. 8. These envelopes are similar to those formed by encysting diatoms (22). we did not amplify 18S rDNA consistently from adult reproductive tracts (Fig. 3I) indicate possible oviductal transmission. from vernal pools (35) has been described.net. along with a positive control bacterial 16S rRNA-targeted probe (EUB338) (39) and negative control bacterial sense probe (NON338) (40).. This may account for larger vacuole sizes of invading algae. Behavioral stimuli. reveal that this symbiosis is a true mutualism (6. 8. We designed an oligonucleotide probe speciﬁc for Oophila 18S rRNA (5′-TCTCTCAAGGTGCTGGCGA-3′) based on the regions of low RNA folding complexity in eukaryotes (38). 12). cut in 100-μm sections in 1% agarose (wt/vol) on a Vibratome 1000 (Automatic Tissue Sectioning Systems). Canada under permits from The Wildlife Division of the Nova Scotia Department of Natural Resources. V(D)J recombination in B and T cells. stained with saturated (2%) uranyl acetate in 50% (vol/vol) ethanol. However. Animal procedures followed Dalhousie University Committee on Laboratory Animals protocol (09-029). 26). Although our data are consistent with a process of vertical transmission. These are often revealed through incongruent phylogenetic topologies between host and symbiont populations (20). 9). Printz) (19). we do not know whether these envelopes are independently derived or whether they represent a lineage of encysted algal cells. Nova Scotia. However. the embryonic association of the Ambystoma–alga symbiosis may preclude an adaptive immune response that would otherwise remove invading algal cells. Despite their structural similarity. S2). comparisons between Ambystoma-Oophila populations could reveal the extent of vertical or horizontal acquisition in different salamander populations and test the controversial (6) species designation “Oophila amblystomatis” (Lambert ex. but unlike the association of Chlorella sp. 11). the material beneﬁt of algal symbionts has been controversial. Additionally. does not occur until 6–8 wk after fertilization in the axolotl Ambystoma mexicanum (4). and development of both host and symbiont. Color images were acquired on a Nikon AZ0 stereo dissecting microscope. as indicated by RAG-1 protein in the thymus. Fluorescence and TEM. The number of potential antibodies used in adult salamander antigen response is restricted (36). 3B). postﬁxed in 2% osmium tetroxide in 0. Most intracellular algae were in direct contact with the host cytoplasm and not enclosed in a distinct vesicular membrane. and the similar encystment of some intracellular algae (Fig. However. Fluorescent in Situ Hybridization. which may account for their remarkable regenerative abilities and acceptance of allo. 2% glutaraldehyde in 0. neither the process of environmental acquisition (15) nor free-living Oophila sp. More reﬁned microelectrode studies have since shown that intracapsular algae do produce a net increase of oxygen (11. Several intracellular algae were enclosed in a thickened envelope. which may instigate algal proliferation (8. were purchased from biomers. clutches grown in alga-free tap water failed to grow algae in comparison with those raised in pond water (6. 3 A and B). algae primarily enter salamander embryos through the blastopore before the formation of a patent stomodeum and therefore before active feeding is possible. which are contained in a symbiosomal membrane. well after the initial invasion of algae in A. dehydrated in an ethanol series. such as gradients of nitrogenous waste.4). Individual embryos were anesthetized in tricaine methanesulfonate (pH 7.nih. this process is unlikely to be the dominant mode of algal acquisition. maculatum. As suggested in the Introduction. and embedded in Spurr’s resin (Structure Probe). the encysted algae on the innermost egg capsule (Fig. 15) or oocyte (15) rinses. The horse radish peroxidase-conjugated Oophila-speciﬁc probe. if these algae are following a nitrogenous waste gradient. However. One-way ANOVAs of area measurements with Games-Howell post hoc tests were run on SPSS (version 18). and cellular integration is often a result of partial digestive assimilation (17. in A.
1 M Tris (pH 7. 2 × 5 min in 0. 8. Sigma).5 μL/mL acetic anhydride. New York). Each slide was covered with 150 μL hybridization buffer [900 mM NaCl.1 M triethanolamine (pH 7. 34. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2. ACKNOWLEDGMENTS. phylogenetic. Karakashian S (1970) Morphological plasticity and the evolution of algal symbionts. Sunderland. Amann R (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of ﬂuorescently labeled oligonucleotide probes. Mol Biol Evol 28:699–706. Cheng L. Nat Rev Microbiol 8:218–230. 27. Vol 23. New Haven. diffusion and oxygen production by algae. and Evolution (Sinauer Associates. 1. Bachmann M. diversity.5). Zelzer E. A case of symbiosis. Fundamental Immunology. MA). Archaea. Oophila-speciﬁc 18S rDNA ampliﬁcation in salamander tissues. ed Goff L (Cambridge Univ Press. Biol Bull Mar Biol Lab Woods Hole 115:483–489. Freshwater Algae of North America. Hutchison V. Can J Zool 86:1289–1298. R.H. et al. and in vivo time-lapse microscopy. 35. Proc Natl Acad Sci USA 105: 17867–17871. with additional observations on the hypophysis. Prochloron sp. 9. Rumpho M. Gatz J (1973) Algal entry into the eggs of Ambystoma maculatum. Epel D. Can J Zool 64: 1586–1588.A.Salamander tissues were ﬁxed in 4% paraformaldehyde (PFA) overnight. Pardy R (1983) Phycozoans. Hoober J (Springer. Slides were covered with 150 μL of ampliﬁcation reagent and incubated for 45 min in the dark (25 °C) before sequential washes (20 min and 15 min) with TNT buffer (46 °C) and 1. 37. and environmental studies in microbiology. Muscatine L. Phycozoologists? Algal Symbiosis: A Continuum of Interaction Strategies. Harrison R (1969) Harrison stages and description of normal development of the spotted salamander. Dordrecht. Hammen C. Bhavsar AP. Shudert E (2003) Nonmotile coccoid and colonial green algae. 15 min in 0. Nature 449:827–834. van Doorn WG. Pinder A.1018259108 Kerney et al. et al. pp 253–307. Hirose E. Mech Dev 106:97–106. 46 °C] and an additional 15 min in wash buffer (46 °C). Q J Micro Sci N S 115:483–489. Krumholz L. Du Pasquier L (2009) Evolution of the immune system. 33. et al.H is supported in part by Natural Sciences and Engineering Research Council (NSERC) Grant 298366-2009 to Alastair Simpson.). 13. Flajnik M. 15. Lippincott Williams & Wilkins.01% SDS. and 5 mM EDTA. Philadelphia). Germany). Perspect Plant Ecol Evol Syst 8:23–43. Gilbert PW (1944) The alga-egg relationship in Ambystoma maculatum. Sci World J 6(Suppl 1):1–11. Ann N Y Acad Sci 175:474–487. 31. Medicine. 30. in a colonial ascidian. Pool RR. Sheath RG (Academic Press. 17. Trench RK (1975) Symbiosis of algae and invertebrates: Aspects of the symbiont surface and the host-symbiont interface. Prehybridization steps followed Zelzer et al. and the appendages and skeleton of the head. 10. and 10% (wt/vol) dextran sulfate] and included an unlabeled helper oligonucleotide (5′-GTCATCAAAAGAACGCTCGCC-3′) (42) and HRP-conjugated probe (0. Chesneau A.5 mg/mL DAPI counterstaining (5 min.. and National Science Foundation Grant MCB-0848083 (to R. 29. SI Materials and Methods provides additional methods pertaining to the online supporting material. is supported by the Tula Foundation. eds Engler A. Worden AZ. Phycozoology. phylogenetic reconstructions. Appl Environ Microbiol 66:3603–3607. eds Wise R. 24. Ambystoma punctatum (Linn). Glöckner FO.1073/pnas.K. Hoegh-Guldberg O (2006) The evolutionary history of Symbiodinium and scleractinium hosts—symbiosis. and Robin Kodner. J Exp Biol 210:2430–2435. 28. Slides were cover-slipped with 150 μL of AF1 (Citiﬂuor) and sealed with nail polish. Spiegelaar N (2008) Embryonic motility and hatching success of Ambystoma maculatum are inﬂuenced by a symbiotic alga. Charlemagne J (1987) Antibody diversity in amphibians. Princeton). 32.0. Vol 3. Gilbert PW (1942) Observations on the eggs of Ambystoma maculatum with especial reference to the green algae found within the egg envelopes. Valls JH. pp 5–18. 42. 36. John Gilhen. 39. David Hewitt.1% Tween-20). Bull N Y Zool Soc 30:51–56. Loram JE. Lane CE. (2008) Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. J Bacteriol 172:762–770. New York). 6. Biol Cell 100:503–521. Binder BJ (2000) In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. J Herpetol 7: 137–138. E. Douglas A (2010) The Symbiotic Habit (Princeton Univ Press. Lee J (2006) The kleptoplast. Burkholder J. Ecology 25:366–369. 10 min in 5 μg/mL proteinase-K in PBS (37 °C). and John Archibald. and air-dried for 30 min. Michelle Leger and Yana Eglit provided useful comments on an earlier draft. Carter D.02 M HCl. Orr H (1888) Note on the development of amphibians. (2003) In situ accessibility of small-subunit rRNA of members of the domains Bacteria. pp 1–463. Organization and Development of the Embryo. Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative. Trends Ecol Evol 23:268–275. Invertebr Biol 115: 343–348. Printz H (1928) Chlorophyceae. 0.0–8. 11. Ecology 23:215–227. Life Sci 22:1463–1468. 38. Archibald JM (2008) The eukaryotic tree of life: Endosymbiosis takes its TOL.5). Appl Environ Microbiol 69:1748–1758. 6th Ed. eds Wehr TD. Venn AA. Trench R (1979) The cell biology of plant-animal symbiosis. Oophila amblystomatis. 7. Neff AW (2006) Limb regeneration in amphibians: Immunological considerations.5). 6502 | www. Trans Am Microsc Soc 94: 450–469. following the manu- facturer’s instructions for tetramethylrhodamine or cyanine-3 tyramide in 40% (wt/vol) dextran sulfate. Slides were washed 2 × 5 min in PBS in between each step up to the triethanolamine treatment.K. Dodge J (1973) The Fine Structure of Algal Cells (Academic Press. Supporting Methods. 40. 3. 26. Breder R (1927) The courtship of the spotted salamander. Wetzel R (1985) Symbiosis between salamander eggs and green algae: Microelectrode measurements inside eggs demonstrate effect of photosynthesis on oxygen concentration. Buchner P (1965) Endosymbiosis of Animals with Plant Microorganisms (John Wiley. 19.15 ng/mL ﬁnal concentration each). Rumpho ME. New York). Appl Environ Microbiol 66:284–289. 1% blocking reagent. Tattersall G. Mescher AL. Guttman JA. Slides were hybridized for 3 h in a humidity chamber (46 °C). 16. Douglas AE (2008) Photosynthetic symbioses in animals. et al.pnas. with rRNA-targeted peptide nucleic acid probes. 2. Prantl K (W. 25% (vol/vol) formamide.K. rinsed in sterile phosphate buffered saline (PBS). Behrens S.H.). (2008) Transgenesis procedures in Xenopus. dehydrated in a graded ethanol series. Engelmann. Carlton R. Eur J Immunol 17:421–424. 159 mM NaCl. 41. Annu Rev Plant Physiol 30: 485–531. Lewin R (1996) Intracellular symbiosis of a photosynthetic prokaryote.).4dinitrophenyl antibody responses. Slides were then reﬁxed in 4% PFA for 5 min. pp 451–473. Manhart JR. CT). Ambystoma maculatum and the alga. 0. (2011) Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes. ed Paul W (Wolters Kluwer. an NSERC grant (to B. 21. 25. 23. J Exp Biol 197:17–30. then 2 × 5 min in 0. Die natürlichen Pﬂanzenfamilien [The Natural Plant Families]. (2001) Tissue speciﬁc regulation of VEGF expression during bone development requires Cbfa1/Runx2. Cambridge. The Structure and Function of Plastids (Advances in Photosynthesis and Respiration). 5.1 M triethanolamine solution containing 2. Bulgheresi S (2010) A complex journey: Transmission of microbial symbionts. 150 mM NaCl. (41) for mRNA in situ hybridizations and included rehydration into PTw (PBS with 0.K. Yoshimoto K (2010) Role of chloroplasts and other plastids in ageing and death of plants and animals: A tale of Vishnu and Shiva.074% Tween 20] for 15 min (25 °C). 25 °C). Finlay BB (2007) Manipulation of host-cell pathways by bacterial pathogens. A. 4. 14.org/cgi/doi/10. This research was funded by the American Association of Anatomists (R. and the effect of climate change. Goff LJ. Gilbert SF (2008) Ecological Developmental Biology: Integrating Epigenetics. followed by a brief rinse in prewarmed wash buffer [20 mM Tris (pH 7. 20. Dastoor FP. Friet S (1994) Oxygen transport in egg masses of the amphibians Rana sylvatica and Ambystoma maculatum: Convection. 10 min in 3% H2O2. Fuchs BM. UK). is an American Association of Anatomists Scholar. Chisholm SW. Ageing Res Rev 9: 117–130. Stein JR (1978) Ammonia: Basis for algal symbiosis in salamander egg masses. Slides were soaked in TNT buffer [0. Stat M. 12. A TSA plus ﬂuorescence kit (PerkinElmer) was used for signal ampliﬁcation. These methods include ampliﬁcation and sequencing of nuclear 18S and chloroplast 16S rDNA of Oophila. We thank Joe Martinez. Wulf J. Mills NE (2007) Intermittent hypoxia in eggs of Ambystoma maculatum: Embryonic development and egg capsule conductance. et al. mouth. Leipzig. and 0. Maruyama T. Hutchison V (1962) Carbon dioxide assimilation in the symbiosis of the salamander Ambystoma maculatum and the algae Oophila amblystomatis. pp 44–66. rinsed 5 × 1 min in PBS. and stored in 100% methanol (−20 °C). 18. Life Sci 1: 527–532.02% SDS. . The Netherlands). 20 mM Tris (pH 7. and Eucarya to Cy3-labeled oligonucleotide probes. Hammen C (1958) Oxygen utilization in the symbiosis of embryos of the salamander. pp 56–124. 22. Slides were covered with paraﬁlm strips during the hybridization and tyramide signal ampliﬁcation (TSA) steps. This work was partially completed in the laboratories of James Hanken. J Exp Bot 59:1069–1080. Amann RI. Bright M. ed Wilens S (Yale Univ Press. Wägele H. chieﬂy concerning the central nervous system. Alastair Simpson.P.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.